WO2003096798A1

WO2003096798A1 - Tetraploid watermelons producing small fruits

Info

Publication number: WO2003096798A1
Application number: PCT/US2003/014849
Authority: WO
Inventors: Xingping Zhang
Original assignee: Syngenta Participations Ag
Priority date: 2002-05-14
Filing date: 2003-05-12
Publication date: 2003-11-27
Also published as: EP1503621A4; IL164927A0; BR0309980A; AU2003243222A1; CN1652680A; CN100539828C; MXPA04011208A; AU2003243222B2; US20060037110A1; EP1503621B1; EP1503621A1; US8742208B2; US20080005814A1; US7652193B2; IL164927A; US7547550B2; US20050034197A1; JP2006507796A

Abstract

Tetraploid watermelon inbreds are disclosed. The invention relates to the seeds and plants of the tetraploid watermelon inbreds, the methods of propagating the tetraploid inbreds through seeds and tissue culture. The invention also relates to methods of producing the triploid seedless watermelon seeds and plants by crossing the tetraploid inbreds with diploid watermelon inbreds, and to the triploid plants produced therefrom.

Description

TETRAPLOID WATERMELONS PRODUCING SMALL FRUITS

FIELD OF THE INVENTION This invention is in the field of watermelon breeding, specifically relating to a tetraploid watermelon used to produce triploid seeds and plants for production of a substantially small sized seedless watermelon fruit.

BACKGROUND OF THE INVENTION Watermelon is an important horticultural crop that accounts for 2% of the world area devoted to vegetable crops. There were 6,024,000 acres of watermelon grown in the world and 187,000 acres of watermelons grown in the United States in 1997. Asia is by far the most important watermelon production site with 72% of the world area and 77% of the world production (FAO Production Yearbook 51, 1998). The estimated annual world watermelon value exceeded $7.6 billion when using the United States average price for 1995-1997. The United States watermelon crop amounted to over 41 million cwt, from over 174,000 harvested acres, and a farm value of over $266 million, accounted for 9.2% of the harvested acres, 10.0% of the production, and 3.5% of the value of the United States fresh vegetable industry in 1999 (USDA Agricultural Statistics 2001). California was the leading state in watermelon farm gate value, exceeded $72 million in 2000, due to high percentage of triploid seedless watermelon grown in California. Seedless watermelon receives well above the average price for seeded watermelons in the market. Triploid seedless watermelon also produces higher yields than the diploid seeded watermelons.

Triploid seedless watermelon is a true FI hybrid between a tetraploid watermelon, as the female parent, and a diploid watermelon, as the male parent (Kihara, H. 1951, Triploid Watermelons, Proceedings of American Society for Horticultural Science, 58:217-230). Regular watermelons, the seeded diploid watermelons, have 22 chromosomes (2N = 2X = 22) in their somatic cells. The tetraploid watermelons have 44 chromosomes (2N = 4X = 44) in their somatic cells. When female flowers of tetraploid plants are cross-pollinated by the male flowers of normal diploid plants, the seeds produced in the fruit of tetraploid plants are triploid seeds. Triploid seeds produce triploid plants. When the triploids plants are grown with the normal diploid plants in the same field, the triploid plants produce fruits that are seedless. The seedless condition in triploid watermelon is the result of the presence of three homologous sets of chromosome per somatic cell rather than the usual two. Cells with three sets of homologous chromosomes are said to be triploid and are designated as 3X. The triploid seedless watermelons have 33 chromosomes (2N = 3X = 33) in their somatic cells. The inability of the triploid zygote to produce normal viable gametes (pollen and egg cells) causes the absence of seeds in triploid fruits. Typically, seedless watermelons contain small edible white ovules, similar to those in immature cucumbers.

Triploid seedless watermelons have been commercially grown in the United States since the late 1980's. The popularity of seedless watermelon has increased since its commercial introduction in the United States. Most of the watermelons produced in California in 2001 were triploid seedless watermelons. Triploid varieties produce higher yields than the diploid seeded varieties, due to more fruit per plant and longer harvest period. The triploid seedless watermelon receives premium prices because of the high quality flesh virtually free of seeds.

The present invention addresses the need for triploid watermelons producing smaller seedless fruits while maintaining high standards of fruit taste and texture. However, when the size of the fruit is reduced, the proportion of the rind in the fruit increases, leaving only little space for the edible flesh. Thus, it is also an objective of the present invention to develop watermelons producing small seedless fruits with high flesh content. The present invention also addresses the need for tetraploid watermelon plants, which can be used as parents for triploid watermelons producing such small seedless fruits.

SUMMARY OF THE INVENTION Accordingly, the present invention provides new and unique tetraploid watermelons. The tetraploid watermelons of the present invention are preferably tetraploid inbred lines and are particularly suitable as parent for triploid hybrids watermelons. The tetraploid watermelons of the present invention produce small fruits with a very thin rind. The fruits of the tetraploid watermelons of the present invention have preferably a high content of soluble solids. Importantly, the tetraploid watermelons of the present invention are capable of transmitting these desirable characterictics to their triploid progeny when crossed with diploid watermelons. Therefore, the present invention also discloses triploid watermelons that produce small-sized fruits virtually free of seeds and having a very thin rind. Such seedless watermelon fruits have the advantage of having more edible flesh with excellent taste and texture when compared to watermelons of comparable size, and are therefore particularly attractive to the consumer.

In a preferred embodiment, a tetraploid watermelon of the present invention produces a fruit weighting about 1.5 kg to about 2.5 kg, preferably about 1.5 kg to about 2 kg, preferably about 2 kg. In another preferred embodiment, the rind of the fruit of a tetraploid watermelon of the present invention is about 4 mm to about 10 mm thick, preferably about 4 mm to about 7 mm thick. In another preferred embodiment, a tetraploid watermelon of the present invention has excellent fruit set. In another preferred embodiment, a tetraploid watermelon of the present invention has high sugar content. In another preferred embodiment, a tetraploid watermelon plant according to the present invention produces seeds weighting about 20 g to about 35 g per 1000 seeds, preferably about 23 g to about 33 g per 1000 seeds, preferably about 31 g per 1000 seeds, in another preferred emdodiment about 25 g per 1000 seeds. In another preferred embodiment, a tetraploid watermelon plant according to the present invention produces up to about 150 seeds per fruit, preferably about 50 to about 150 seeds per fruit, preferably about 70 to about 150 seeds per fruit, more preferably about 80 to about 150 seeds per fruit. In another preferred embodiment, said plant produces about 3-4 fruits per plant. In another preferred embodiment, the fruit of said plant comprises about 12 to about 14 % of soluble solids, preferably about 13%. In another preferred embodiment, the fruit of said plant matures about 24 to about 27 days after flowering.

In another preferred embodiment, a tetraploid watermelon of the present invention includes one or more of the following characteristics: a) produces a fruit that is significantly smaller than the fruit of conventional tetraploids, approximately 1/3 the size of conventional tetraploids, b) displays exceptional fruit set ability, c) produces 3-4 fruits per plant in a greenhouse setting, d) matures 7 to 10 days earlier than regular tetraploids as measured from flower to fruit maturity, e) the seed is much smaller in size than the seed of conventional tetraploids, f) the fruit rind is about 1/2 to 1/3 of the regular tetraploids, g) the seed yield is significantly better than most of the tetraploids created with the same technique.

In yet another prefereed embodiment, the present invention relates to a new and unique inbred tetraploid watermelon line, designated 90-4194 and including the characteristic above.

In another preferred embodiment, when a tetraploid watermelon of the present invention is used a parent for production of a triploid watermelon, said triploid watermelon produces a small seedless fruit having a very thin rind. Preferably, the seedless fruits weights about 2 kg to about 3 kg. In another preferred embodiment, the rind of the seedless fruits is about 4 mm to about 13 mm thick, preferably about 7 mm to about 11 mm thick.

In another preferred embodiment, when a tetraploid watermelon of the present invention is used a parent for production of a triploid watermelon, said triploid watermelon including one or more of the following characteristics: a) small fruit size, b) more fruit per plant, c) fruits having higher sugar content, d) thin rind, usually 1/2 or 1/3 the thickness of conventional triploids, e) the triploid fruits maturing about 7-10 days earlier than currently available triploid seedless watermelon fruit, f) the triploids of the invention are more tolerant to hollow heart compared to currently available triploid seedless watermelon fruits, g) excellent fruit set ability, h) soluble solid content above about 11%, preferably about 11% to about 13%.

The present invention also includes the seeds of a tetraploid watermelon line of the present invention. The present invention also includes pollen, ovule, fruit of a tetraploid watermelon of the present invention, and methods of producing hybrid triploid watermelon seeds using a tetraploid watermelon line of the present invention. This invention further includes triploid hybrid watermelon seeds produced by crossing a tetraploid watermelon line of the present invention with a diploid watermelon line, and plants obtained from such seeds, including fruits thereof. The present invention further includes methods of producing seeds having all the genetics of a tetraploid watermelon line of the present invention, methods of developing new inbred tetraploid lines using a tetraploid watermelon line of the present invention, and methods for producing a tetraploid watermelon line using a diploid watermelon line.

The present invention further provides:

A method of producing triploid hybrid watermelon seed by crossing a tetraploid watermelon plant of the present invention with a diploid watermelon plant. Preferably, said tetraploid plant is the female parent. Preferably, said diploid plant is the male parent. A triploid hybrid watermelon seed produced by a method above.

The present invention further provides: A method to produce triploid hybrid watermelon seed comprising the steps of: a) planting in pollinating proximity seed of a tetraploid watermelon plant according to the present invention and seed of a diploid watermelon plant; b) planting seed of said diploid watermelon plant 7 to 10 days earlier than seed of said tetraploid watermelon plant; c) cultivating watermelon plants resulting from said seeds until said plants bear second female flowers; d) covering the female flowers of said tetraploid watermelon plant on the afternoon immediately prior to the day said female flowers open; e) collecting the male flowers of said diploid watermelon plant in early morning on the day said female flowers open; f) pollinating said female flowers with said male flowers collected in step e); and g) harvesting seeds produced from said female flowers pollinated in step f).

The present invention further provides: A method to produce triploid watermelon seed according comprising the steps of: a) planting seed of a tetraploid watermelon plant according to the present invention and seed of a diploid watermelon plant in a field in a pattern of 2 rows of tetraploid watermelon plants to 1 row diploid watermelon plants; b) pruning said tetraploid watermelon plant to approximately 2-3 branches per plant; c) emasculating substantially all the male flowers and male flower buds on the plants of said tetraploid watermelon plant; d) allowing pollination of the female flowers of said tetraploid watermelon plant with the pollen of said diploid watermelon plant; e) marking the female flowers pollinated during the emasculating period; and f) harvesting the seed from the marked fruit of the tetraploid watermelon plant.

The present invention further provides: A method of producing tetraploid watermelon seed comprising: a) growing a tetraploid watermelon plant according to the present invention; b) allowing self-pollination of said tetraploid watermelon plant; c) obtaining a fruit from a plant of step b); d) extracting tetraploid watermelon seed from said fruit. Preferably, the method further comprises washing and drying said seed.

The present invention further provides:

A method of developing a tetraploid watermelon line in a watermelon plant breeding program using plant breeding techniques, which include employing a watermelon plant, or its parts, as a source of plant breeding material, comprising: obtaining the watermelon plant, or its parts, of the present invention as a source of breeding material.

The present invention further provides:

A method for producing a tetraploid watermelon plant comprising: a) obtaining a diploid watermelon line producing a small-sized fruit with a thin rind, b) subjecting plants of said diploid watermelon line to tetraploid conversion to produce tetraploid watermelon plants, c) selecting a tetraploid watermelon plant obtained in step b), wherein said tetraploid watermelon plant produces a fruit weighting about 1.5 kg to about 2.5 kg and having a rind with a thickness of about 4 mm to about 10 mm. In a preferred embodiment, the tefraploid watermelon produces a fruit weighting about 1.5 kg to about 2 kg, preferably about 2 kg. In another prefened embodiment, the rind of the fruit of a tefraploid watermelon of the present invention is about 4 mm to about 7 mm thick. In another preferred embodiment, the tetraploid watermelon line produces seeds weighting about 20 g to about 35 g per 1000 seeds. In another prefened embodiment, the tetraploid watermelon line produces up to about 150 seeds per fruit, preferably about 50 to about 150 seeds per fruit, preferably about 70 to about 150 seeds per fruit, more preferably about 80 to about 150 seeds per fruit. In another preferred embodiment, the tetraploid watermelon line produces about 3-4 fruits per plant. In another prefened embodiment, the fruit of said tetraploid watermelon line comprises about 12 to about 14 % of soluble solids. In another prefened embodiment, the fruit of said tetraploid watermelon line matures about 24 to about 27 days after flowering. In another prefened embodiment, the watermelon tetraploid line comprises any one of the characteristics disclosed herein. fri another prefened embodiment, said diploid watermelon line is a diploid inbred watermelon line, preferably diploid inbred watermelon line HD or a diploid inbred watermelon line having the characteristics of diploid inbred watermelon line HD. In another prefened embodiment, the diploid line is converted to a tetraploid line using an oryzalin protocol. fri another prefened embodiment, the method further comprises self-pollinating a tetraploid watermelon plant selected in step c) above. In another prefened embodiment, the method further comprises self-pollinating a tetraploid watermelon plant selected in step c) above until no variation is observed in the progeny.

The present invention further provides: A tetraploid watermelon line obtainable by a method disclosed immediately above, including pollen, ovule, fruit of said tetraploid watermelon line.

DETAILED DESCRIPTION OF THE INVENTION In commercial production of triploid watermelon seed, tetraploid and diploid parental lines are planted in the same field. Cross-pollination between the tetraploid line, the female parental line, and the diploid line, the male parental line, are accomplished by either hand or bee pollination. Triploid watermelon seeds are produced only in melons of tefraploid plants that are fertilized with pollen of diploid plants. All commercially grown seeded watermelons are diploid; therefore, there are many diploid lines for use as diploid parents. The major limitation to development of seedless watermelon varieties lies in the availability of useful tetraploid parental lines. In general, it is difficult to predict the performance of a tefraploid watermelon from based on the characteristics of the diploid. A good diploid watermelon does not necessarily produce a good tetraploid. Morever, a tefraploid with good horticultural traits does not necessarily produce good triploid watermelon hybrids.

Tetraploid watermelon lines are developed by doubling the chromosomes of regular diploid watermelon lines. Chromosome doubling was first accomplished with the toxic alkaloid colchicme by applying colchicine to the growing point of new emerged watermelon seedlings. Tissue culture methods have also been developed by Zhang, X.P., B.B. Rhodes, H.T. Skorupska, W.C. Bridges, 1995, Generating Tetraploid Watermelon Using Colchicine in Vitro, G. Lester & J. Dunlap et al. (eds.), Cucurbitaceae' 94: 134-139. Dinitroanilines have been used to double chromosome numbers, and their effectiveness has previously been compared with crops other than watermelon. Li et al (Li, Ying, J.F. Whitesides, B. Rhodes, 1999, In vitro generation of tetraploid watermelon with two different dinitroanilines and colchicines, Cucurbit Genetics Cooperative Rpt 22:38-40) compared in vitro chromosome doubling effectiveness using colchicine and the dinitroanilines, ethalfluralin (N-ethyl-N-2- methyl-2-propenyl)-2,6-dinitro-4-(trifluoromethyl) benzamne), and oryzalm (3,5-dinitro-N4, N4-dipropylsulfanilamide) and concluded that either ethalfluralin or oryzalin was preferable to colchicine.

Several treatment methods are used to induce tetraploids from diploids using the chemicals mentioned above. One method is to treat the seed before sowing. The seed are soaked in clean water for 5-6 hrs and then the seed are soaked in either colchicine solution (0.2%) or dinitroanilines (e.g. 35 μM oryzalin) for 24 hrs. The seed are briefly rinsed before sowing. Dry seed can also be directly soaked in the chemical solution without pre-soaking in the water. This treatment is simple to do and is a good method to use if one has no restriction of seed supply. The treatment usually reduces the germination and emergence. The second method is to treat the new emerged seedling. The diploid inbreds are sown in greenhouse in seedling flats. The soil temperature is kept at 29 - 31°C for rapid and uniform germination. One drop of colchicine (0.1%) or dinitroanilines (e.g. 35 μM oryzalin) solution is added to the shoot apex between the cotyledons as soon as the seedling has emerged from soil. The colchicine solution is applied to the growing point in the morning or evening for three consecutive days. We get good chromosome doubling from one application of oryzalin. Another method is to treat the shoot apex of germinated seed after which the germinated seed is planted into soil. The seeds are germinated in an incubator at 30°C. When the radicals are about 2 cm long, the portion above the hypocotyls of germinated seeds is immersed upside down into colchicine (0.1%) or dinifroaniline solution (35 μM oryzalin) for 10-15 hrs at 30 °C in an incubator. The treatment should be conducted in a high humidity chamber or box to assure that the radicals/roots are not desiccated. The seeds are then washed and planted in the soil. The last two methods, although more tedious to use, usually give better recovery of tefraploid events as the root system is not affected by the treatment.

The next step is to develop tetraploid lines from individual converting events. The selected tefraploid individuals based on morphological expression are self-pollinated and the resulting seeds are planted in the next generation as lines. These lines are again self-pollinated and compared for fertility and horticultural traits. Only the desirable lines are selected if there is difference among these lines. Desirable lines may be bulk harvested if there is no variation within the line and among selected lines. Further seed increases may be conducted in an isolation block. Mass selection may be conducted for this increase in the isolation plot and thereafter. Fertility of the tetraploid may be improved in subsequent generations.

The use of tissue culture to propagate tetraploid watermelon plants is further exemplified in Adelberg, J.W., B.B. Rhodes, Micropropagation from zygotic tissue of watermelon, C.E. Thomas (ed.) Proc. of the Cucurbitaceae 89: Evaluation and enhancement of cucurbit germplasm, Charleston S.C., USA; and Zhang et al., Shoot regeneration from immature cotyledon of watermelon, Cucurbit Genetics Coop. 17:111-115 (1994).

Crossing two different tetraploids and then going through recombination breeding can also result in new tetraploid lines. A longer breeding period is required to develop a stable tetraploid line using this approach. This is due to the larger number of combinations and the fewer seed that tetraploids produce. However, some breeders make good progress by taking this approach. Because meiosis is sometimes inegular in autotefraploids, diploids and aneuploids do occur in their offspring. The leaves, flowers and pollen grains of tefraploids are morphologically distinct from diploids (Zhang, X.P., B.B. Rhodes, H.T. Skorupska, W.C. Bridges, 1995, Generating Tetraploid Watermelon Using Colchicine in Vitro, G. Lester & J. Dunlap et al. (eds.), Cucurbitaceae' 94: 134-139). Tetraploids also have a different number of chloroplasts in the guard cells (Compton, M.E., DJ. Gray and G.W. Elmstrom. 1996, Identification of tetraploid regenerants from cotyledons of diploid watermelon cultures in vitro, Euphytica 87:165-172). These morphological traits can help breeder to eliminate the diploids and aneuploids occurring in the tetraploid population during sexual propagation. Triploid seeds are cunently produced using two methods, the bee-pollination method and the hand-pollination method. In the United States, the bee-pollination method is used to produce triploid watermelon seed. Almost all of the United States triploid watermelon seed production is located in Northern California. The production fields are typically planted in a ratio of 2 rows of tetraploid female line and 1 row of diploid male line. All the male flower buds are manually removed from the female tetraploid plants. This process is known as de- budding. The female flowers are open-pollinated by bees. The fruit set during the de-budding period are marked and harvested for triploid hybrid seed. Male buds are manually removed from tetraploid female vines throughout the pollination season. If a male sterile tetraploid line is available, workers can easily remove the male fertile plants in the tetraploid female row with much less time and efforts. All the fruit set on the male-sterile tetraploid plants can be harvested for hybrid triploid seed. When the marked male-sterile system is used, seed producer can insure that no female off-types exist in the female tetraploid line and the hybrid triploid seed (Zhang, X.P. and B.B. Rhodes, 2000, Method using male sterility and a marker to produce hybrid seeds andplants; U.S. Patent 6,018,101). Hand-pollination is mainly used to produce triploid watermelon seed in areas where isolation is not available and several triploid hybrids are produced in the same field block. Inbred male parent line is sown 7-10 days earlier than inbred female tetraploid parent line. The male parent is usually located outside of the crossing block. Approximately four to ten tetraploid female plants per male plant are planted to insure adequate pollination. The male parent is carefully checked for its uniformity before male flowers are collected. Any off-types that can be recognized based on plant morphology and ovary characteristics are removed. Pollination starts when the second female flowers of the tetraploid female parent are ready to flower. The female flower buds of the tetraploid female parent line are identified and covered with paper cups or small paper bags before they bloom the next morning. Male flowers of the diploid male parent line are collected in the early morning before the visit of bees or other pollination insects to the flowers. The covered female flower buds are then uncovered and pollinated using the collected fresh male flowers. The pollinated female flowers are then recovered and marked. The open-pollinated fruits on the female parent plants are removed periodically to insure the development of hand-pollinated fruits. Male plants are removed from the field after pollination is complete to insure that only fruit from female parents are harvested.

Development of tetraploid lines of the present invention

A problem regularly observed with the conversion of diploid watermelon lines to tefraploid lines is that the fertility rate of the tetraploid line is dramatically reduced. This leads to a low fruit set and is often coπelated with late fruit setting and late maturity of the fruit. Consequently, seeds production of tetraploid watermelons is inefficient and expensive, and tetraploid watermelons are generally not good parents for triploid hybrid seeds production. An objective of the present invention is therefore to produce tetraploid watermelons overcoming these fertility problems, while having the advantageous characteristics disclosed in the instant application and being able to transmit these characteristics to their triploid progeny in full or at least in part.

Diploid inbred watermelon line HD, a proprietary Syngenta line used as a parental line for the production of diploid seeded watermelon hybrids, was identified as a promising candidate for tetraploid conversion. Diploid line HD produces small-sized seeded fruits with a thin rind, and has excellent fruit set. Moreover, diploid line HD produces fruits with high sugar content and has a firm flesh. Diploid watermelon line HD was subjected to tetraploid conversion.

Inbred 90-4194 was developed in northern California by converting diploid inbred HD to a tetraploid watermelon. The conversion from diploid (2X) to tefraploid (4X) was accomplished using an oryzalin protocol comprising the following steps: In November of 1999, seeds of HD were seeded in a 50-cell plastic seedling tray in the greenhouse. One drop of 35 μM oryzalin was added to the meristem tip between 2 cotyledons each of the newly emerged seedlings. Treatment of all the seedlings with oryzalin was finished about 10 days after sowing. Seedlings were watered and fertilized periodically.

In late December of 1999, putative tetraploids were fransplanted into 2-gallon pots filled with Pro-Mix BX soil-less soil in the greenhouse. During the course of plant development, diploid (not converted) plants and branches were removed based on leaf morphology and male flower characteristics. Following is the chronological order of development of tetraploid watermelon lines, for example inbred line 90-4194:

Generation Season / Year Description

To Spring 2000 At the seedling transplant stage, 72 putative tetraploids were transplanted into 2-gallon pots in greenhouse. Non-converted plants and branches were identified based on leaf morphology and male flower characteristics, and were removed. About 20 tetraploids were identified and only the female flowers from true tetraploid plant/branches were self-pollinated. At full fruit maturity, fruit with large blossom end scars (2-3 times that of its diploid version) were harvested and examined for fertility as suggested by number of seed per fruit. Four individual selections 4XHD-1, -2, -3, and -4 and one bulk selection 4XHD-B were made to plant the T] generation.

Ti Summer 2000 4XHD-1 and 4XHD-2 were planted in the greenhouse for further selection and seed increase. 4XHD-3 and 4XHD-4 were planted in the field for field observation and seed increase. 4XHD-B was planted in the crossing block in the field to make triploid hybrids. 4XHD-2 was not as good as 4XHD-1 at the seedling stage and was discarded. 42 plants of 4XHD-1 were grown to maturity in the greenhouse. All the seeds of 4XHD-1 were bulk-harvested and labeled as 4XHD-1-B as no variation was observed in this line. No variation was observed within and between 4XHD-3 and 4XHD-4. Therefore seeds were also bulk-harvested and labeled as 4XHD-3/4. Fall 2000 Five triploid hybrids derived from 4XHD-B were evaluated in

Florida. Three hybrids were unique and promising triploid hybrids. T₂ Spring 2001 About 700 plants of 4XHD-1-B were planted in a plastic greenhouse for generation advance and seed increase. Hand pollination was conducted. No variation was observed. All the fruits are uniform and true to type. Seeds were bulk-harvested and named as 90-4194. T₃ Summer 2001 About 3500 plants of 90-494 were fransplanted to a one-acre isolation plot for stock seed increase using bee-pollination. 1200 plants of 90-4194 were transplanted into 2 net covered cages for foundation seed increase by hand pollination. No variation was observed from cage and field plantings. The breeding process is finished and seeds harvested serve as foundation and stock seed. The unique characteristics of a tetraploid watermelon line of the present invention, for example inbred 90-4194, are described as follows. The values indicated below are for plants grown under summer conditions in Northern California. Variations may be observed when the plants are grown under substantially different conditions.

A tefraploid watermelon line of the present invention is a very early maturing tetraploid watermelon. For example, fruit of 90-4194 matured 24-27 days after flowering under summer conditions in Northern California. Other commercial tefraploids take at least 35 days under the same conditions.

The fruit of a tetraploid watermelon line of the present invention is very small, about 1.5 kg to about 2.5 kg, preferably about 1.5 kg to about 2 kg, preferably about 2 kg, only approximately 'Λ -1/3 the size of the tetraploids commercially used. The fruit shape is round and skin color is light green with green pencil lines. The flesh is firm and red with refractometer % soluble solids (mostly sugars) of about 12% to about 14%, preferably about 13%.

The seeds of a tetraploid watermelon line of the present invention are small, preferably about 20 g to about 35 g per 1000 seeds, preferably about 23 g to about 33 g per 1000 seeds, preferably about 31 g per 1000 seeds, in another prefened emdodiment about 25 g per 1000 seeds, approximately ^l - 1/3 the weight of commercially used tetraploids.

A tetraploid line of the present invention has a good seeds yield. The seeds yield is high when the tetraploid line is self-pollinated or when the tetraploid line is pollinated by a diploid male parent to produce triploid seeds. In a prefened embodiment, a tetraploid line of the present invention produces up to about 150 seeds per fruit, preferably about 50 to about 150 seeds per fruit, preferably about 70 to about 150 seeds per fruit, more preferably about 80 to about 150 seeds per fruit. In another prefened embodiment, a tetraploid line of the present invention produces an average of about 80 seeds per fruit. The seed color is medium brown. The rind is very thin, about 4 mm to about 10 mm, preferably about 4 mm to about 7 mm, approximately VT. - 1/3 the thickness of commercially used tetraploids. This thin rind splits easily under dry conditions with great relative humidity fluctuations.

A tetraploid line of the present invention has excellent fruit setting ability under poor environmental conditions. When 90-4194 was grown in the plastic greenhouses without supplement lights in the early spring of 2001 at Syngenta's Woodland research station with other 23 diploid and 1 tetraploid breeding lines, it was the only watermelon line setting fruits normally. Most of the watermelon lines did not produce any fruit due to poor growing conditions.

A tetraploid line of the present invention can produce multiple fruits per plant. For example, one plant of 90-4194 can bear as many as 4 fruits per plant. A tetraploid line of the present invention can also produce fruit in a long period of time. For example, 90-4194 produced 2 crops from one planting during stock seed increase in the summer of 2001 Northern California. After harvest of first fruit set 90-4194 produced a second fruit set with excellent seed yield.

A tetraploid line of the present invention produces triploid hybrids with fruit size about 3 kg or smaller, preferably about 2.5 kg or smaller. All the traditional triploid hybrids have much bigger fruits, most are above 6 kg. Preferably, fruits of the triploid hybrids further comprise one of the following characteristics: small fruit size, more fruit per plant (preferably 3-4 fruits per plant), higher sugar content, thin rind (the rind of the seedless fruits is about 4 mm to about 13 mm, preferably about 7 mm to about 11 mm), usually 1/2 or 1/3 the thickness of conventional triploids, the triploid fruit mature about 7-10 days earlier than regular triploid seedless watermelon fruit, the triploids developed from the invention are more tolerant to hollow heart, excellent fruit set ability, soluble solid content above about 11%, preferably about 11% to about 13%.

A tetraploid watermelon inbred of the present invention can be multiplied through vegetative propagation and sexual propagation. The vegetative propagation can be done preferably via shoot proliferation and then rooting in tissue culture. The detailed methods were described by Zhang et al. (Zhang, X.P., B.B. Rhodes, H.T. Skorupska, W.C. Bridges. 1995. Generating Tetraploid Watermelon Using Colchicine in Vitro. G. Lester & J. Dunlap et al. (eds.), Cucurbitaceae' 94: 134-139). This method is briefly described as follows: A) Collect shoot-tips and axiliary buds from greenhouse, field or laboratory grown plants of 90- 4194. Rinse the shoot-tips and axillary buds under tape water for 30 minutes to an hour. Sterilize the shoot tips and axillary buds in 10% of household bleach for 8 - 10 minutes. Rinse the shoot-tips and axillary buds three times in sterilized distilled water. B) Culture the shoot- tips and axillary buds on Murashige and Skoog (MS) medium with 10 μM BA for shoot proliferation. Make a subculture every 3-4 weeks. C) Root the proliferated shoot buds on MS medium with 5-10 μM IBA for about 2 weeks. D) Acclimatize the rooted plantlets in a temperature and humidity controlled greenhouse. E) Grow the micropropagated plants in an isolation plot and harvest the seed from these plants.

Tetraploid watermelons are usually sexually propagated through seed. We have successfully propagated inbred 90-4194 in the greenhouse, in a net covered cage, and in the open fields. The seed increase field should be isolated from any other watermelon by at least 1.5 KM, if the seed increase is conducted in the open field. Good pest management and cultural practices should be implemented. Higher (20 - 30% higher than those used for commercial hybrid fruit production) levels of phosphate and potassium fertilize are beneficial for producing high seed yield and good seed quality. Calcium is supplemented for the fields low in calcium to minimize the fruit loss due to fruit splitting. Beehives are placed in the seed production fields to insure good pollination, the key biological event for seed production. Fruit is preferably harvested before fruit split and deterioration. The harvested fruit is then stored under room temperature for a period of time before extracting seed from fruit, to allow further embryo development and seed maturation in the fruit. The seed, after being extracted from the flesh, is thoroughly washed and quickly dried using a forced-air dryer to best maintain the seed viability.

The primary use of tetraploid watermelon is to make triploid hybrid watermelon seeds and plants that produce seedless fruit. The tetraploid line is used as female parent to cross with the diploid watermelon lines, the male parent lines. The creation of a desirable triploid hybrid heavily relies on the performance, especially seed producibility, and the combining ability of the tetraploid parent. A tetraploid watermelon inbred of the present invention, for example line 90-4194, is a novel tefraploid line for creating triploid seedless watermelon hybrids having excellent fruit quality, small fruit size (personal size in the range of 2 - 3 KG), early maturity and excellent fruit setting ability.

It is preferable to select the diploid male parents on the basis of small fruit size, ideally less than 8 lbs, preferably less than 6 lbs. In addition, the skin color and stripe pattern that is desired for the triploid watermelon is selected from the diploid male parent, as the skin color and stripe pattern (non) of a tefraploid of the present invention are recessive to all other skin color and stripe patterns. Diploid male parents are also selected for their small seed size to reduce the size of the white seed ovules in the triploid fruit. Most tetraploids are very difficult or fail to produce triploid seed when they are grown in greenhouse. However, inbred 90-4194 produced triploid seed with good seed yield in the greenhouse. Inbred 90-4194 also produced triploid seed in the field. Tefraploid watermelon lines of the present invention have very good combining ability in creating triploid hybrids. For example, with limited exploration, 5 desirable triploid hybrids were created by crossing inbred 90-4194 with different diploid male lines.

Several methods can be used to produce triploid seeds from a tetraploid inbred line of the present invention, once the proper combination is determined. Two commonly used methods are described here with respect to line 90-4194 as example. Variations to these methods can be made according to actual production situation. Hand-pollination method. This is the most often used method for producing triploid seed from 90-4194. The inbred tetraploid female parent 90-4194 and the inbred diploid male parent line are planted in the same field. The inbred male parent is planted 7-10 day earlier than the female parent 90-4194 to insure adequate pollen supply at the pollination time. The male parent and female parent 90-4194 are planted in the ratio of 1 male parent to 4-10 female parents. The diploid male parent may be planted at the top of the field for efficient male flower collection during pollination. Pollination is started when the second female flower on the tetraploid female parent 90-4194 is ready to flower. Female flower buds that are ready to open the next day are identified, covered with paper cups or small paper bags that prevent bee or any other insect visit of the female flowers, and marked with any kind of material that can be easily seen the next morning. This process is best done in the afternoon. The male flowers of the diploid male parent are collected in the early morning before they are open and visited by pollinating insects. The covered female flowers of the tefraploid female parent, which have opened, are un-covered and pollinated with the collected fresh male flowers of the diploid male parent, starting as soon as the male flower sheds pollen. The pollinated female flowers are again covered after pollination to prevent bees and any other insects visit. The pollinated female flowers are also marked. Only the marked fruits are harvested for exfracting triploid hybrid seed.

Bee-pollination method. Using the bee-pollination method, the tetraploid female parent 90-4194 and the diploid male parent are usually planted in a ratio of 2 rows tetraploid parent to 1 row male parent. The female tetraploid plants are pruned to 2-3 branches. All the male flower buds on the female tefraploid parent plants are removed manually, (the de-budding process), during the pollination season on a daily basis. Beehives are placed in the field for transfer of pollen by bees from the male parent to the female flowers of the female parent. Fruits set during this de-budding time are marked. Only the marked fruits are harvested for extracting hybrid triploid seed. The fruit of inbred 90-4194 split easily when they reach maturity, due to its extremely thin rind. This can be a serious problem when the seed production is conducted under conditions of dramatic humidity fluctuations. Thus, the fruit is harvested right before the splitting, and stored in shade or under vine for a period of 2-3 weeks. The rind becomes durable when the fruit is removed from the vine, and the embryos continue to develop in the harvested fruit, resulting in better seed quality.

According to the invention, tefraploid inbreds are used as parental lines to develop new tetraploid lines. The unique desirable traits a tetraploid watermelon line of the present invention, for example line 90-4194, make it also very useful as a parental line in the development of new tefraploid inbreds. Such line can be used as either female or male parent to cross with another inbred or hybrid tefraploid to develop new tetraploid inbreds.

Example 1 : Description of the tefraploid inbred 90-4194 Fruit: round, small

Area of best adaptation: most areas Emergence of anthesis: 5 days earlier than the commercial tefraploid variety

90-4231 Pollination to maturity: 7 days earlier than the commercial tefraploid variety

90-4231

Ploidy: tefraploid

Cotyledon: flat

Sex: monoecious

Number of Main Stems: 3 at crown

Number of flowers at first fruit set: 18 staminate

4 pistillate

Stem: round, pubescent, 7 mm diameter at second node

Jhternode length 7 cm Flower at first fruit set: staminate 3 cm across pistillate 3 cm across color yellow

Mature fruit size: round, 15 cm long, 15 cm diameter at midsection, 2 kg average weight, smooth, light green rind with pencil lines, mottle/net

Rind: brittle, 4 mm thick blossom end, 7 mm thick sides Flesh: crisp, fine with little fiber, medium red, 13% soluble solids of juice, no hollow heart, placental separation, or transverse crack

Seed: 7 mm long, 5 mm wide, 2 mm thick, index (length ÷ width x 10) is 14, 31 gm per 1000 seed, 80 seed per fruit, dark brown

Sunburn: resistant

In contrast, the commercial tetraploid line 90-4231 has a fruit size of approximately 6-7 kg, and expresses some hollow heart, placental separation, and transverse crack.

In summary, the uniqueness of the inbred tefraploid watermelon line 90-4194 of the invention includes the following: a) The tetraploid 90-4194 produces a fruit that is significantly smaller than the fruit of conventional tefraploids, approximately 1/3 the size of conventional tetraploids; b) The tefraploid 90-4194 displays exceptional fruit set ability. In Honduras, the tetraploid 90-4194 is able to produce 3 crops (harvest of triploid seed from the tetraploid plant) from 1 planting, whereas conventional tetraploid plants allow for only 1 such harvest. Likewise, in California, Peru, and Chile, the 90-4194 of the invention produces 2 crops from 1 planting, c) The tetraploid 90-4194 produces 3-4 fruits per plant in a greenhouse setting, whereas conventional tefraploids typically produce on the average of less than 1 fruit per plant, d) The tefraploid 90-4194 matures 7 to 10 days earlier than regular tetraploids as measured from flower to fruit maturity, e) The tefraploid seed of 90-4194 of the invention is much smaller in size than the seed of conventional tefraploids. Twelve to 17 seeds of conventional tefraploid watermelon make a gram. In contrast, 35 to 39 90-4194 seeds of current invention make a gram, f) The fruit rind of 90-4194 is about 1/2 to 1/3 of the regular tetraploids. g) The seed yield of the 90-4194 tefraploid plant is significantly better than most of the tetraploids created with the same technique. 90-4194 produces as many as 150 triploid seed per fruit when pollinated by diploid male parent. Most of the recently developed tefraploid watermelons typically produce about 10 to 75 triploid seed per fruit when pollinated by diploid male parents .

Example 2: Triploid watermelon fruits

The triploids watermelon fruit grown from triploid seed produced by a tetraploid plant of the invention display commercially important and novel traits. For example, the uniqueness of the triploids produced by 90-4194 according to the invention, compared to conventional seedless watermelon, includes a) small fruit size, b) more fruit per plant, c) higher sugar content (the SSC), (see Table 1 below), d) thin rind, usually 1/2 or 1/3 the thickness of conventional triploids, e) the triploid fruit mature about 7- 10 days earlier than regular triploid seedless watermelon fruit, f) the triploids developed from the tefraploid 90-4194 of the invention are more tolerant to hollow heart, a common defect of triploid seedless watermelon. Hollow heart on triploid seedless watermelon RWT 8123 and RWT 8124 has not been observed. RWT 8123 and RWT 8124 are triploids produced by the tetraploid 90-4194. and g) excellent fruit set ability.

Table 1 shows the average weight (Avg. Wt), the number of fruit per plant (Frt/plant) and the amount of soluble solids (mostly sugar) in the fruit flesh (SSC%). Tri-X-313 is a standard sized commercial triploid seedless watermelon, using a conventional tefraploid watermelon as the female parent. RWT8123 and RWT8124 are triploid seedless watermelons using the tefraploid 90-4194 watermelon of the invention as the female parent. The male diploid parents of the Tri-X-313, RWT8123, and RWT8124 lines were chosen for their significantly different phenotypes.

Table 1:

Table 2 below shows that the fruit of RWT 8124 (see above) is much small than regular triploid watermelon and that the fruit set of RWT8124, as expressed by number of fruit per plant, is much higher than that of commercially available seedless watermelons.

Table 2:

In accordance with the method of the invention, it is preferable to select the diploid male parents on the basis of small fruit size, ideally less than 8 lbs. In addition, the skin color and stripe pattern that is desired for the triploid watermelon is selected from the diploid male parent, as the skin color and stripe pattern (non) of 90-4194 are recessive to all other skin color and stripe patterns. Diploid male parents are also selected for their small seed size to reduce the size of the white seed ovules in the triploid fruit.

Although the foregoing invention has been described in some detail in this document, it will be obvious that changes and modification may be practiced within the scope of he invention, as limited only by the scope of the appended claims.

DEPOSIT Applicants have made a deposit of at least 2500 seeds of diploid inbred watermelon line

HD [HD(2X90-4194)] with the American Type Culture Collection (ATCC), Manassas, Virginia, 20110-2209 U.S.A., ATCC Deposit No: PTA-5146. This deposit of the diploid inbred watermelon line HD will be maintained in the ATCC depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the effective life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicants have satisfied all the requirements of 37 C.F.R. §§1.801-1.809, including providing an indication of the viability of the sample. Applicants impose no restrictions on the availability of the deposited material from the ATCC; however, Applicants have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicants do not waive any infringement of its rights granted under this patent or under the Plant Variety Protection Act (7 USC 2321 et seq.).

The foregoing invention has been described in detail by way of illustration and example for purposes of clarity and understanding. However, it will be obvious that certain changes and modifications such as single gene modifications and mutations, somaclonal variants, variant individuals selected from large populations of the plants of the instant inbred and the like may be practiced within the scope of the invention, as limited only by the scope of the appended claims.

All references cited herein are incorporated by reference in the application in their entireties.

Claims

What is claimed is:

1) A tetraploid watennelon plant producing a fruit weighting about 1.5 kg to about 2.5 kg and having a rind with a thickness of about 4 mm to about 10 mm. 2) The tetraploid watermelon plant according to claim 1, producing a fruit weighting about 1.5 kg to about 2 kg and having a rind with a thickness of about 4 mm to about 7 mm.

3) The tetraploid watermelon plant according to claim 1, producing seeds weighting about 20 g to about 35 g per 1000 seeds.

4) The tetraploid watermelon plant according to claim 1, producing seeds weighting about 31 g per 1000 seeds.

5) The tetraploid watermelon plant according to claim 1, producing up to about 150 seeds per fruit.

6) The tetraploid watermelon plant according to claim 1 , producing about 50 to about 150 seeds per fruit.

7) The tetraploid watermelon plant according to claim 1, producing about 70 to about 150 seeds per fruit.

8) The tetraploid watermelon plant according to claim 1, producing about 80 to about 150 seeds per fruit.

9) The tetraploid watermelon plant according to claim 1 , wherein said plant produces about 3-4 fruits per plant.

10) The tetraploid watermelon plant according to claim 1 , wherein said fruit comprises about 12 to about 14 % of soluble solids.

11) The tetraploid watermelon plant according to claim 1, wherein said fruit comprises about 13 % of soluble solids.

12) The tetraploid watermelon plant according to claim 1 , wherein the fruit of said plant matures about 24 to about 27 days after flowering. 13) Seed of the tetraploid watermelon plant according to any one of claims 1-12. 14) Pollen of the tefraploid watermelon plant according to any one of claims 1-12. 15) An ovule of the tefraploid watermelon plant according to any one of claims 1-12.

16) Fruit of the tetraploid watermelon plant according to any one of claims 1-12.

17) A method of producing triploid hybrid watermelon seed by crossing a tetraploid watermelon plant according to any one of claims 1-12 with a diploid watermelon plant. 18) The method of claim 17, wherein said tefraploid plant is the female parent.

19) The method of claim 17, wherein said diploid plant is the male parent.

20) A triploid hybrid watermelon seed obtainable by the method of claim 17.

21) A method to produce triploid hybrid watermelon seed comprising the steps of: a) planting in pollinating proximity seed of a tetraploid watermelon plant according to any one of claims 1-12 and seed of a diploid watermelon plant; b) planting seed of said diploid watermelon plant 7 to 10 days earlier than seed of said tetraploid watermelon plant; c) cultivating watermelon plants resulting from said seeds until said plants bear second female flowers; d) covering the female flowers of said tetraploid watermelon plant on the afternoon immediately prior to the day said female flowers open; e) collecting the male flowers of said diploid watermelon plant in early morning on the day said female flowers open; f) pollinating said female flowers with said male flowers collected in step e); and g) harvesting seeds produced from said female flowers pollinated in step f).

22) A method to produce triploid watermelon seed comprising the steps of : a) planting seed of a tetraploid watermelon plant according to any one of claims 1-12 and seed of a diploid watermelon plant in a field in a pattern of 2 rows of tetraploid watermelon plants to 1 row of diploid watermelon plants; b) pruning said tefraploid watermelon plant to approximately 2-3 branches per plant; c) emasculating substantially all the male flowers and male flower buds on the plants of said tetraploid watermelon plant; d) allowing pollination of the female flowers of said tetraploid watermelon plant with the pollen of said diploid watermelon plant; e) marking the female flowers pollinated during the emasculating period; and f) harvesting the seed from the marked fruit of the tefraploid watermelon plant. 23) A method of producing tetraploid watermelon seed comprising: a) growing a tefraploid watermelon plant according to any one of claims 1-12; b) allowing self-pollination of said tetraploid watermelon plant; c) obtaining a fruit from a tefraploid watermelon plant of step b); d) extracting tefraploid watermelon seed from said fruit.

24) The method according to claim 23, further comprising washing and drying said seed.

25) A method for producing a tefraploid watermelon plant comprising: a) obtaining a diploid watermelon line producing a small-sized fruit with a thin rind, b) subjecting plants of said diploid watermelon line to tetraploid conversion to obtain tefraploid watermelon plants, c) selecting a tetraploid watermelon plant obtained in step b), wherein said tetraploid watermelon plant produces a fruit weighting about 1.5 kg to about 2.5 kg and having a rind with a thickness of about 4 mm to about 10 mm. 26) The method according to claim 25, wherein the tetraploid watermelon plant selected in step c) produces seeds weighting about 20 g to about 35 g per 1000 seeds.

27) The method according to claim 25, wherein the tetraploid watermelon plant selected in step c) produces up to about 150 seeds per fruit.

28) The method according to claim 25, wherein the tetraploid watermelon plant selected in step c) produces about 50 to about 150 seeds per fruit.

29) The method according to claim 25, wherein the tetraploid watermelon plant selected in step c) produces about 70 to about 150 seeds per fruit.

30) The method according to claim 25, wherein the tefraploid watermelon plant selected in step c) produces about 80 to about 150 seeds per fruit. 31 ) The method according to claim 25, wherein the tetraploid watermelon plant selected in step c) produces about 3-4 fruits per plant.

32) The method according to claim 25, wherein the fruit of said tefraploid watermelon plant selected in step c) comprises about 12 to about 14 % of soluble solids.

33) The method according to claim 25, wherein the fruit of said tefraploid watermelon plant selected in step c) matures about 24 to about 27 days after flowering. 34) The method according to claim 25, wherein said diploid watermelon line is diploid inbred watermelon line HD or a diploid inbred watermelon line having the characteristics of diploid inbred watermelon line HD.

35) The method according to claim 25, wherein said step b) comprises subjecting said plants to an oryzalin treatment.

36) The method according to any one of claims 25, further comprising: d) self-pollinating a tefraploid watermelon plant selected in step c).

37) The method according to any one of claims 25, further comprising: d) self-pollinating a tefraploid watermelon plant selected in step c) until no variation is observed in the progeny.

38) A tefraploid watermelon line obtainable by the method of any one of claims 25-36.

39) Seed of the tefraploid watermelon line according to claim 37.

40) Pollen of the tetraploid watermelon line according to claim 37.

41) An ovule of the tetraploid watermelon line according to claim 37. 42) Fruit of the tetraploid watermelon line according to claim 37.

43) Use of a tefraploid watermelon line according to any one of claims 1-12 to produce a hybrid triploid watermelon.

Applicant's or agent's International applicatfόnNo file reference 70028_WOPCT

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13bis)

A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 21 _ jj^e 15-1 / _

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10801 University Blvd., Manassas, VA20110-2209 USA

Date of deposit Accession Number

April 22, 2003 PTA-5146

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Form PCT RO/₁₃4 (Julyl998)